Evaluation of a novel lateral flow immunochromatographic assay for the rapid detection of KPC, NDM, IMP, VIM and OXA-48 carbapenemases in Gram-negatives

The dissemination of carbapenemase-producing (CP) Gram-negatives is a matter of great clinical concern given the major role of these pathogens as causes of nosocomial infections. The production of carbapenemases and ESBLs has become a signature for difficult-to-treat infections (DTIs) because the spectrum of available therapies is drastically reduced.¹ The rapid and effective detection of antibiotic-resistant bacteria is a critical step for antibiotic stewardship and infection control.^1,2 Despite technological improvements, the identification of pathogenic bacteria, as well as the detection of antibiotic resistance, remains complex and time-consuming, with time to results often above 24–48 h or associated with very high costs, such as multiplex PCRs.³

The most widespread carbapenemases in Enterobacterales belong to Ambler class A (mostly KPC enzymes), MBLs (Ambler class B) of NDM, VIM and IMP type, and carbapenem-hydrolysing Ambler class D enzymes of OXA-48 type.^3,4 Phenotypic approaches such as antimicrobial susceptibility, even though cheap, require bacterial growth (>24 h), and are often difficult to interpret as CPs may remain susceptible to carbapenems thus requiring confirmatory testing.³ Biochemical tests, based on β-lactam hydrolysis, have shown their usefulness as confirmatory tests but lack sensitivity, and are difficult to interpret as β-lactam hydrolysis may be weak in certain genetic backgrounds.^3,4 Molecular detection of relevant β-lactamase genes is fast, sensitive and specific, but requires specialized equipment and skills, and is relatively expensive.³ Lateral flow immunoassay (LFIA)-based diagnostics have proved to be useful they are easy (no training), rapid (<15 min) and reliable (nearly 100% sensitivity and specificity) confirmatory tests for detection of the five main carbapenemases in Gram-negatives.⁴ They perfectly match the WHO ASSURED criteria [Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment-free, Deliverable (long shelf life at room temperature)].^2,4 In numerous publications, LFIAs have also been used successfully directly on clinical specimens such as positive blood cultures, urine samples and rectal swabs.^4,5

In a recent study, Bernabeu et al.⁶ compared the performances of three commercially available LFIAs able to detect the ‘big five’ carbapenemases (KPC, OXA-48-like, NDM, VIM and IMP) [NG-Test CARBA 5 (NG-Biotech, Guipry-Messac, France), RESIST-5 K.O.N.V.I (Coris-BioConcept, Gembloux, Belgium) and CP-5 (ERA-Bio, Pittsford, USA)] using a collection of well-characterized Enterobacterales. NG-Test CARBA 5 showed the best performance, especially with IMP variants, which are the most challenging carbapenemases, due to a great variability.⁷ Here, a novel LFIA, the Carbapenem Resistant Enterobacteriaceae Test Kit (CRE-LFA) (Dynamiker, Tianjin, China) was compared with NG-TEST CARBA 5.

Ninety-one well-characterized Enterobacterales with reduced susceptibility to carbapenems were tested, including 86 CPEs (6 VIM producers, 24 OXA-48 producers, 13 KPC producers, 7 NDM-like producers, 14 IMP producers, 13 multiple carbapenemase producers and 9 minor carbapenemase producers) and 5 non-CPEs (Table 1).

All isolates were grown on Mueller–Hinton agar (Bio-Rad, Marnes-la-Coquette, France). Briefly, for NG-Test CARBA 5, three single colonies were touched using a plastic spreader, resuspended in five drops of lysis buffer by vortexing and 100 μL was directly added to the sampling hole of the test cassette as recommended by the manufacturer (NG-Biotech) (Figure S1a, available as Supplementary data at JAC Online). For CRE-LFA, a 1 µL inoculating loopful of bacteria was resuspended in 300 μL of extraction buffer, vortexed for 10 s and incubated for 10 min at room temperature, after which 200 μL was added to the sampling hole of the test cassette (Figure S1A). For both tests, the time to positivity of the test bands and their intensity after the 15 min migration were recorded using a gold colour card scale, (Figure S1b; NG Biotech). The tests of both assays were performed on the same day from the same bacterial culture. Readings were performed at 15 min by two independent persons blinded regarding the tested isolates.

The specificity was 100% (95% CI = 73.24%–100%) for both assays, as no false positive results were observed for none of the targeted carbapenemases (Table 1).

The sensitivity was 97.40% (75/77, 95% CI = 90.07%–99.55%) for NG-Test CARBA 5 and 81.82% (63/77, 95% CI = 71.03%–89.36%) for CRE-LFA (Table 1). While NG-Test CARBA 5 missed 2/15 non-carbapenemase KPC variants (namely KPC-31 and KPC-33), CRE-LFA failed to detect 8/24 OXA-48 variants, 2/7 NDM variants, 2/14 IMP variants and 2/13 multiple carbapenemases producers. (Table 1). CRE-LFA failed to detect NDM-1 in Morganella morganii and an NDM-1/VIM-2 in Enterobacter cloacae. These isolates are known to express these enzymes at a low level. In addition, NDM signals were overall weak using the CRE-LFA test (Figures S1c and S2). In addition, while NG-Test CARBA 5 detected all IMP-producing isolates, CRE-LFA failed to detect 3/13 isolates and gave low intensity bands for 6 additional strains (Table 1, Figure S2). Of note, the accurate detection of IMP might be crucial in endemic regions (e.g. South-East Asia, Australia, Taiwan, Japan), but also in Gram-negative non-fermenters, where these enzymes are more prevalent than in Enterobacterales.⁷

While NG-Test CARBA 5 detected all OXA-48 variants, CRE-LFA failed to detect OXA-48 variants displaying mutations in the β5-β6 loop (Figure S1c). This is a major drawback as some of these variants, such as OXA-244 and OXA-232, are currently detected all over the world with increasing prevalence, and others display both carbapenem and expanded-spectrum hydrolytic activity (OXA-517, OXA-793).^8,9,10 In addition, for a few isolates, low intensities were recorded for CRE-LFA (Table 1; Figure S2). NG-Test CARBA 5 detected OXA-163 and OXA-405, which are considered non-carbapenemase OXA-48 variants (Table 1).¹⁰

Overall, both tests shared common features, including rapidity and simplicity, but NG-Test CARBA 5 exhibited better performance compared with the CRE-LFA tests, mainly for the accurate detection of NDM producers, IMP and difficult-to-detect OXA-48 variants (such as OXA-244, OXA-232). The overall migration on CRE-LFA is faster than on NG-Test CARBA 5 (Figure S2), but this increased migration speed is likely at the expense of lower band intensities and thus non-detection, as the contact between the antibodies and antigens on the strip is reduced. These low intensity bands might be an issue for accurate reading, as seen with CRE-LFA (Figure S1c5 and c6).

Notably, and unlike CP-5 and RESIST-5 K.O.N.V.I, which require two different detection cassettes,⁶ NG-Test CARBA 5 and CRE-LFIA use only one cassette, which in ecologically driven times is highly relevant to reduce plastic waste.

Acknowledgements

We are grateful to NG Biotech for providing NG-Test CARBA 5 for free.

Funding

This study was supported by internal funding.

Transparency declarations

None to declare.

Supplementary data

Figures S1 and S2 are available as Supplementary data at JAC Online.

References